Cardiolipin (CL) is one of three structural lipids found in the membrane fraction of the gram-negative bacteria, Escherichia coli. The CL content of E. coli varies widely depending upon culture conditions. The function of CL and the factors that regulate its synthesis are not known. Circumstantial evidence suggests that under certain culture conditions E. coli can grow normally despite an inability to synthesize CL. Recent investigations performed in this laboratory indicate that cells with a lesion in a gene responsible for CL synthesis, the cls gene, are resistant to the glycerol 3-phosphate analog, 3,4-dihydroxybutyl 1-phosphonate (DBP). Furthermore, such cells are unable to grow at elevated pH. These two observations provide an experimental basis for exploring the function of CL and the factors that regulate its synthesis. Advantage will be taken of gene "libraries" and genetic techniques to isolate a plasmid that contains the cls gene. CL synthetase will be purified from a strain bearing this plasmid and will be characterized to determine the factors that regulate enzyme activity. MudI will be used to interrupt the cls gene and to construct strains in which the lac operon is fused to the cls promoter. These strains will be used to evaluate whether CL is a dispensable lipid and to study the factors that regulate CL formation at the genetic level. The inability of cls strains to grow at elevated pH's suggests that CL may play a role in Na+/H+ antiporter function. This possibility will be investigated. Phenotypic revertants of cls strains will be isolated and characterized to discover compensatory biochemical processes. Additional DBP - resistant mutants will be isolated to determine whether lesions in genes other than cls lead to drug resistance. Strains that are resistant to high concentrations of DBP will be isolated in the hope of uncovering new information concerning the function of anionic phosphoglycerides.